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Laboratory of Molecular Immuneregulation, SAIC-FCRDC, National Cancer Institute, Frederick, Maryland
Correspondence: Kathrin Muegge, SAIC-FCRDC, Laboratory of Molecular Immunoregulation, National Cancer Institute, Bldg. 560, Rm. 31-45, Frederick, MD 21702-1201. E-mail: MACROBUTTON HtmlResAnchor muegge{at}mail.ncifcrf.gov
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ABSTRACT |
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 TOP ABSTRACT MECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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c and Jak3, is required
for successful recombination at the TCR- locus. IL-7 signaling
controls the initiation phase of V(D)J recombination by controlling
access of the V(D)J recombinase to the locus. In the absence of IL-7,
the TCR- locus is methylated and packaged in a repressed form of
chromatin consisting of hypoacetylated histones. IL-7 signaling likely
increases the acetylation state of the nucleosomal core histones
resulting in an "open" form of chromatin. This opening leads to a
higher accessibility for the transcription machinery and increased
accessibility of the Rag heterodimer that performs the cleavage of
DNA.
Key Words: lymphoid development • TCR- locus • immunoglobulin • transposon • Rag-1/Rag-2
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MECHANISM OF V(D)J RECOMBINATION |
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TOPABSTRACTMECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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Two mechanisms guarantee the unlimited diversity of our immunoreceptor gene repertoire: the recombination event and the modification of the broken DNA ends before ligation. Taming the ancient transposon led to the creation of the unique process of V(D)J recombination, enabling us to encounter the antigenic diversity of our environment.
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CONTROL OF V(D)J RECOMBINATION IS REQUIRED FOR LYMPHOID DEVELOPMENT |
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TOPABSTRACTMECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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One level of control lies in rag gene expression (reviewed in [6 ]). Another level of control was first postulated by Alt and colleagues (reviewed in [7 ]) to explain cell-type specificity, time specificity, and locus specificity of V(D)J recombination. For example, T and B cells express rag-1 and rag-2 genes and share the same recognition sequences positioned adjacent to their immune-receptor genes. However, only T cells rearrange their TCR genes, and B cells, their Ig genes, fully. Thus cell-type specific changes are thought to regulate access for the Rag dimer to the TCR or Ig loci.
Thus, this raises the question as to the identity of the signals that control the process of V(D)J recombination and the molecular mechanism that regulates accessibility for Rag-mediated cleavage at a specific locus.
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INTERLEUKIN (IL)-7 SIGNALING IS REQUIRED FOR NORMAL LYMPHOID DEVELOPMENT |
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TOPABSTRACTMECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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IL-7 is expressed primarily in thymic, epithelial cells or bone marrow, stromal cells, the sites were T and B development, and V(D)J recombination occurs. Therefore, the in vitro studies suggested IL-7 as a candidate for regulation of this important step in early lymphoid development.
It is now well established that IL-7 plays a key role in lymphoid
development. Mice that are deficient in the IL-7R
chain, first
demonstrated by Peschon and colleagues [18
], show a
severe block in lymphoid development. T-cell precursors in the thymus
of IL-7R-/- mice arrest at the earliest stage of T-cell development
(CD4-CD8-CD44+CD25-).
At this early, T1 stage, V(D)J recombination is initiated usually, as
we have shown previously [19
]. Also, deletion of IL-7
itself or other components of the IL-7 signal-transduction pathway
(such as c and Jak3) leads to severe immune deficiencies
in mice [20
21
22
23
24
]. That IL-7 signal transduction is also
important in humans was first demonstrated by Leonard and colleagues
[25
], identifying mutations in the c
chain as the cause of X-linked, severe combined immunodeficiency (SCID)
in humans. Later, mutations in Jak3 gene or aberrant
regulations of the IL-7R chain have been shown to be responsible for
human immune deficiencies (reviewed in [26
]). Thus, IL-7
is crucial for lymphoid development in mice or men.
However, the effects of IL-7 in vivo are complex and at
least partially trophic (i.e., IL-7 affects the survival of lymphoid
precursor cells) and partially based on a direct effect on the process
of V(D)J recombination itself (reviewed in [27
,
28
]). Consequently, immune deficiencies as they occur in
IL-7R-deficient mice can be restored partially by introduction of a
bcl-2 transgene (substituting for the trophic requirement of
IL-7) or are restored partially by introduction of a rearranged TCR
transgene [17
, 29
].
We and others have addressed the question of how IL-7 affects V(D)J recombination in vivo and have demonstrated that IL-7 alters accessibility for the V(D)J recombinase specifically.
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IL-7 IS REQUIRED FOR TCR- GENE REARRANGEMENT
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TOPABSTRACTMECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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chain
show a severe deficiency of gene rearrangement at the three productive
TCR- loci. This defect is truly dependent on IL-7 itself and not on
TSLP, another stromal, cell-derived cytokine
[33
]. Although TSLP and IL-7 share the IL-7R chain,
the c chain and Jak3 are unique to the IL-7
signal-transduction pathway [34
]. Mice that are
deficient in the latter two signal-transduction components, as we have
shown, also lack TCR- chain rearrangement [32
]. The
suppression of TCR- rearrangement is not the only recombination
defect as a result of the absence of IL-7 signaling: IL-7R-/- mice
fail to recombine their Ig heavy-chain locus efficiently
[35
], and IL-7-/- mice show a delayed onset in TCR-ß
chain rearrangement [36
]. Thus, multiple sites are
affected in their capacity to recombine in the absence of IL-7
signaling.
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chain rearrangement could explain the lack of
TCR-
T cells in IL-7R-/- mice because successful
recombination is a prerequisite for T-cell survival and development.
Alternatively, an inhibition of T-cell development could be the
reason for undetectable TCR- rearrangement. The following findings
argue against this second possibility: T1 cells, in the earliest stage
of thymic, T-cell development and the stage at which IL-7R-/-
thymocytes are arrested, initiate rearrangement at the TCR- and -ß
loci at the same time, as we demonstrated recently [19
].
As a result, most ß T cells have a rearranged TCR- locus.
Furthermore, mice that lack any T cells as a result of a
deletion of the TCR- chain, show normal levels of TCR- chain
rearrangement [31
]. Thus, the lack of TCR-
rearrangement in IL-7R-/- mice is not lineage-dependent but is a
result of a defect in recombination. In further support,
IL-7R-deficient mice that are bred onto the C57BL/6J strain are
leaky in ß T-cell development. However, we could demonstrate that
these mice have no detectable TCR- chain rearrangement, although the
requirement for IL-7 to promote pro-T-cell survival is less stringent
on the C57 background [19
]. Finally, Ikuta and
colleagues [3
] have demonstrated that IL-7R-/- mice
expressing a bcl-2 transgene also show no evidence of
TCR- chain rearrangement (Table 1). These results indicate that the
lack of TCR- rearrangement in the absence of IL-7 signaling is
because of an effect on V(D)J recombination and not secondary to an
effect on survival. |
IL-7 CONTROLS INITIATION OF Rag-MEDIATED CLEAVAGE |
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TOPABSTRACTMECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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locus was
demonstrated first by a failure to detect completed, ligated,
recombination products by Southern or polymerase chain reaction (PCR)
analysis. These techniques do not determine which step of V(D)J
recombination was inhibited in the absence of IL-7 signaling: the
initiation of cleavage or the processing and ligation of the
chromosomal ends. This latter possibility had been demonstrated
recently to occur as a consequence of deletion of the TCR-ß enhancer
in mice [37
]. Thus, we searched for the presence of
recombination intermediates in IL-7R-/- thymocytes using
ligation-mediated PCR analysis that allows for detection of broken DNA
ends. The failure to detect any recombination intermediates indicated
that IL-7R-deficient mice do not initiate cleavage at the TCR-
locus [32
]. This result suggests that IL-7 signaling
leads to an alteration of accessibility for Rag-mediated cleavage at
the locus rather than controlling subsequent steps of recombination
such as ligation. In support of this interpretation, we also found a
reduction in germ-line transcripts at the TCR- locus
[32
]. Similarly, a lack of Ig heavy-chain, germ-line
transcripts has been shown in IL-7R-/- mice [35
].
Germ-line transcripts are correlated closely with the recombination
event. Usually, they precede rearrangement briefly and are thought to
indicate an open-chromatin state.
To test directly whether IL-7 controls chromatin accessibility, we used
the Rag-mediated cleavage assay developed by Schlissel and colleagues
[38
]. Originally, their work established that cell-type
specificity of V(D)J recombination is dependent on alteration of
target-site accessibility. Nuclei derived from IL-7R-/- thymocytes
were exposed to recombinant or purified Rag proteins in
vitro, and the chromatin was tested for the initiation of the
cleavage reaction. Thymocytes of IL-7R-deficient mice show a
suppression of recombination intermediates, indicating a reduced access
of Rag proteins [19
]. These results establish that IL-7
signaling controls chromatin accessibility for Rag-mediated cleavage at
the TCR-gg locus.
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IL-7 CONTROLS CHROMATIN ACCESSIBILITY FOR V(D)J RECOMBINATION |
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TOPABSTRACTMECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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locus. DNA
methylation is associated closely with heterochromatin, a
nontranscribed form of chromatin. Using restriction enzymes that are
sensitive to DNA methylation, we have demonstrated by Southern analysis
that the TCR- locus is methylated in the absence of the IL-7 signal
[32
]. Similarly, it was shown recently that the TCR-ß
locus is methylated in mice with a deletion of the constant region
enhancer, a region that is required for successful V(D)J recombination
[39
]. Furthermore, methylation of recombination plasmid
substrates [40
] or episomal substrates
[41
] inhibits the efficiency of V(D)J recombination.
Thus, DNA methylation is associated with inhibition of V(D)J
recombination.
How does methylation of the TCR- locus lead to a suppressed form of
chromatin, and how can IL-7 signaling overcome this suppression? Bird
and colleagues [42
] revealed part of the mechanism by
which DNA methylation results in repressed chromatin and a reduction in
the transcriptional process. They demonstrated that methylated DNA
binds to methyl-DNA-binding proteins, such as MecP2, which then
associates with histone acetylases rendering histones unacetylated. The
unacetylated nucleosomes, formed by the histone core, are associated
with a transcriptionally suppressed form of chromatin. Addition of a
specific, histone-deacetylase inhibitor, such as Trichostatin A (TSA),
can de-repress transcription by elevation of histone-acetylation
levels. Thus, TSA can overcome the suppression of transcription caused
by methylation.
We were able to circumvent the need for IL-7 signaling in V(D)J
recombination by TSA. Thymocytes from IL-7R-/- mice cultured in
the presence of an increasing dose of TSA were able to recombine
successfully at their TCR- locus [32
]. Similarly, it
was shown recently that TSA can overcome the suppression of TCR-ß
recombination caused by deletion of the ß constant region enhancer
[39
], or V
rearrangement can be induced in a
pre-B-cell line [43
]. Furthermore, a block of V(D)J
recombination caused by deletion of regulatory sequences in the TCR-ß
or - genes correlates with low levels of histone-3 acetylation at
the specific loci [39
, 44
]. These findings
suggest that histone acetylation is an important prerequisite for
accessible chromatin, and IL-7 can modulate histone acetylation
specifically at the TCR- locus for V(D)J recombination.
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CONCLUSION AND UNRESOLVED QUESTIONS |
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TOPABSTRACTMECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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locus. Based on the findings of our
laboratory and others, we derive the following model for the molecular
mechanism of IL-7 effects (Table 1)
: IL-7 signaling leads to activation
of specific transcription factors binding to regulatory sequences in
the TCR- locus (Fig. 1)
. These regulatory sites may include recently
identified transcriptional-response elements such as the
constant-region enhancer, the J- promoter, or the human serum
albumin (HSA) binding site located between the variable regions
[3
4
5
]. The binding leads to recruitment of associated
histone acetylases at the TCR- locus. Specific histone acetylation
will lead to an "accessible" form of chromatin correlating with
unmethylated DNA and possibly leading to further modifications of
chromatin including repositioning of nucleosomes and disruption of
higher-order chromatin structure. This opening of chromatin will allow
access of the transcriptional machinery and the appearance of sterile
transcripts. The open form of chromatin will also permit access of the
Rag dimer forming recognition synapsis and initiating the first step of
V(D)J recombination, the site-specific cleavage. The effect of histone acetylation on altering access for the Rag dimer is still unresolved. It has been shown that the core histones in the nucleosome are inhibitory for Rag-mediated cleavage in vitro [45 , 46 ]. However, acetylation of the N-termini of the histones does not relieve this repression in vitro [43 , 45 ]. Thus, histone acetylation may rather affect higher-order chromatin structure in vivo. It is also possible that the histone acetylation in vitro does not mimic the complex acetylation pattern in vivo (involving at least eight different lysine residues at histone 3 or 4) or that other histone modifications such as phosphorylation, methylation, or poly-adenosine 5'-diphosphate (ADP)-ribosylation also contribute to modulation of Rag accessibility. Another possibility is that histone-acetylated N-termini of the histones serve as flags to attract SNF/complexes. Those large SNF complexes can disrupt nucleosomal arrays in vitro and cause sliding of nucleosomes along DNA, thus allowing increased access of other DNA-binding factors. These and other possibilities require future studies.
Currently, it is not known how the IL-7 signal transmits from the
membrane to the nucleus and how a histone acetylase is targeted
specifically to the TCR- locus. The transcription factor Stat5,
translocating to the nucleus in response to IL-7, is a candidate gene
for specific IL-7 signal transduction. Stat5 is not activated in
IL-7R-/- thymi or pre-B cells. Stat5 can complex via the Nmi adaptor
protein with the histone acetylase CBP/p300 [47
].
Furthermore, Stat5 has been shown to bind to the J- promoter, and
there are several other potential Stat5 binding sites in the TCR-
locus [3
]. Thus, Stat5 would be a good candidate to
target histone-acetylase activity, specifically to the TCR- locus.
In support of this model, Ikuta and colleagues [3
] have
demonstrated recently that Stat5 can substitute for IL-7 signaling,
because a Stat5 transgene can restore TCR--chain
rearrangement in IL-7R-/- mice. However, mice that are deficient
in Stat5a and b have been shown to have normal lymphoid development
[48
, 49
]. This argues against Stat5
(although able to substitute for IL-7 signaling), serving as a mediator
for histone acetylation in vivo.
Revealing the molecular mechanism whereby IL-7 controls accessibility
specifically for V(D)J recombination at the TCR- locus may
ultimately provide insights to many cytokine-regulated processes
involving alteration of chromatin structure.
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ACKNOWLEDGEMENTS |
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Received December 13, 2000; accepted January 19, 2001.
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REFERENCES |
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TOPABSTRACTMECHANISM OF V(D)J RECOMBINATIONCONTROL OF V(D)J RECOMBINATION...INTERLEUKIN (IL)-7 SIGNALING IS...IL-7 IS REQUIRED FOR...IL-7 CONTROLS INITIATION OF...IL-7 CONTROLS CHROMATIN...CONCLUSION AND UNRESOLVED...REFERENCES |
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locus by Stat5: implications for the accessibility control by the IL-7 receptor Immunity 11,213-223[Medline]
1 in the murine T-cell receptor locus Proc. Natl. Acad. Sci. USA 88,800-804 variable region genes in murine fetal liver cultures J. Exp. Med. 178,2201-2206 chain-deficient mice Eur. J. Immunol. 27,100-104[Medline]
chain Immunity 2,223-238[Medline]
genes is blocked in interleukin-7 receptor-deficient mice J. Exp. Med. 184,2423-2427 gene rearrangement in interleukin 7 receptor knockout mice Immunol. Lett. 57,9-14[Medline]
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